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The ABCC4 Gene Is Associated with Pyometra in Golden Retriever Dogs Maja Arendt1,2*, Aime Ambrosen3, Tove Fall4, Marcin Kierczak5, Katarina Tengvall2, Jennifer R

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The ABCC4 Gene Is Associated with Pyometra in Golden Retriever Dogs Maja Arendt1,2*, Aime Ambrosen3, Tove Fall4, Marcin Kierczak5, Katarina Tengvall2, Jennifer R www.nature.com/scientificreports OPEN The ABCC4 gene is associated with pyometra in golden retriever dogs Maja Arendt1,2*, Aime Ambrosen3, Tove Fall4, Marcin Kierczak5, Katarina Tengvall2, Jennifer R. S. Meadows2, Åsa Karlsson2, Anne‑Sofe Lagerstedt3, Tomas Bergström7, Göran Andersson7, Kerstin Lindblad‑Toh2,6 & Ragnvi Hagman3* Pyometra is one of the most common diseases in female dogs, presenting as purulent infammation and bacterial infection of the uterus. On average 20% of intact female dogs are afected before 10 years of age, a proportion that varies greatly between breeds (3–66%). The clear breed predisposition suggests that genetic risk factors are involved in disease development. To identify genetic risk factors associated with the disease, we performed a genome‑wide association study (GWAS) in golden retrievers, a breed with increased risk of developing pyometra (risk ratio: 3.3). We applied a mixed model approach comparing 98 cases, and 96 healthy controls and identifed an associated locus on chromosome 22 (p = 1.2 × ­10–6, passing Bonferroni corrected signifcance). This locus contained fve signifcantly associated SNPs positioned within introns of the ATP-binding cassette transporter 4 (ABCC4) gene. This gene encodes a transmembrane transporter that is important for prostaglandin transport. Next generation sequencing and genotyping of cases and controls subsequently identifed four missense SNPs within the ABCC4 gene. One missense SNP at chr22:45,893,198 (p.Met787Val) showed complete linkage disequilibrium with the associated GWAS SNPs suggesting a potential role in disease development. Another locus on chromosome 18 overlapping the TESMIN gene, is also potentially implicated in the development of the disease. Purulent bacterial infection of the uterus (pyometra) is one of the most common diseases of intact female dogs. On average one in fve female dogs are diagnosed with the disease before 10 years of age 1,2. Te proportion of afected bitches diagnosed varies greatly between diferent breeds, i.e. some breeds develop the disease to a much larger extent and at an earlier age than others (from 3% in Finnish spitz’ to 66% in Bernese mountain dogs)1,2. Te clear breed predisposition indicates that genetic risk factors play a role in the pathogenesis. Te golden retriever breed is among the breeds that have increased risk of pyometra (age corrected risk ratio 3.3)2. By 10 years of age, approximately 37% of all intact Swedish female golden retrievers will have been afected by the disease1,2. Pyometra is a potentially life-threatening illness that develops as a consequence of a combination of hormo- nal and bacterial factors. During the luteal phase of the oestrus cycle, high progesterone hormone levels make the uterus susceptible to opportunistic bacterial infections, foremost by Escherichia coli. Infection of the uterus can lead to sepsis and related endotoxemia and organ dysfunctions in severely afected individuals. In addition, circulating infammatory mediators increase3,4. Te treatment of choice is surgical ovariohysterectomy. Non- surgical treatment alternatives are possible in less severe cases, but are frequently associated with disease relapse 5. Diseases of the reproductive organs, such as pyometra, are more commonly diagnosed in Sweden in com- parison to many other countries, where in the latter, most non-breeding female dogs are spayed for reproduction preventive purposes6. Of all Swedish dogs, 90% are insured and 67% are registered in the Swedish Kennel Club 1Faculty of Health and Medical Sciences, Department of Veterinary Clinical Sciences, University of Copenhagen, Copenhagen, Denmark. 2Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden. 3Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden. 4Department of Medical Sciences, Molecular Epidemiology and Science for Life Laboratory, Uppsala University, Uppsala, Sweden. 5Department of Cell and Molecular Biology, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. 6Broad Institute of MIT and Harvard, Cambridge, MA, USA. 7Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden. *email: [email protected]; [email protected] Scientifc Reports | (2021) 11:16647 | https://doi.org/10.1038/s41598-021-95936-1 1 Vol.:(0123456789) www.nature.com/scientificreports/ (SKK), which facilitates identifcation of cases and control dogs suitable for genetic research studies through insurance company databases and the SKK registry7. Here we present an investigation of Swedish golden retrievers to identify genetic risk factors for pyometra using a genome-wide association study approach with a case–control population consisting of clinically well- defned afected and healthy dogs. Results Genome‑wide signifcant locus on chromosome 22 in CanFam 3.1. To identify disease-associated loci, 194 female golden retrievers were genotyped using the 170 k CanineHD BeadChip. Ninety-eight of the dogs were classifed as cases and 96 as controls. Te mean age of onset for the cases was 6.6 years (SD 2.1 years). All controls were intact and > 7 years old with a mean age of 8.6 years (SD 1.4 years). At initial quality control and fltering, 1000 SNPs were removed for low genotyping rate (< 95%) and 72,878 SNPs were removed for having a minor allele frequency of less than 5% leaving 97,468 SNPs for further analysis. No individuals were removed for having a low genotyping rate and the average genotyping rate in the population was 99%. A multidimensional scaling plot was generated showing the frst two dimensions (C1-C2) (Figure S1). No clustering between cases versus controls was noted in the population as a whole. Calculation of relatedness showed that two control dogs were related at sibling level (PI_HAT 0.51, both individuals were kept). A GWAS was performed using EMMAX to account for cryptic relatedness between individuals and population structure8. One genome-wide signifcant locus containing 5 SNPs in complete LD was identifed on chromosome 22 at ~ 45 Mb, p = 1.24 × 10–6, which was below the LD-corrected Bonferroni signifcance threshold calculated as 4.2 × ­10–6 (see QQ-plot and Manhattan plot in Fig. 1a, b). Te QQ-plot did not show evidence of infation (lambda 0.99) with the associated SNPs on chromosome 22 above the dotted line reaching Bonferroni corrected signifcance. Two tentative additional loci were seen in the Manhattan plot (Fig. 1b). A locus on chromosome 18 (top SNP chr18:51,224,157, p = 5.2 × 10 –5), and a locus on chromosome 28 (top SNP chr28: 8,872,257, p = 6.0 × 10 –5). None of these reach Bonferroni corrected signifcance. Conditioning on the top locus. To evaluate if either of the two additional loci represented independent risk factors from the chromosome 22 locus, a conditional genome-wide analysis was performed choosing the genotype of one of the top-associated SNPs (chr22:45,875,420) on chromosome 22 as a covariate. As seen in the QQ plot (Fig. 1c) and Manhattan plot (Fig. 1d), the locus on chromosome 18 shifed ~ 2 Mb but showed a mildly improved p-value leaving it as a suggestive locus (chr18:49,198,998, p = 2.8 × 10–5), whilst the association to the SNP on chromosome 28, located in intron 7 of the SORBS1 gene, disappeared. Te most signifcantly associated SNP on chromosome 18 identifed in this analysis was located in intron 4 of the TESMIN (MTL5) gene (the allele frequency was 0.64 in cases and 0.44 in controls). A closeup of the locus on chromosome 18 including the LD structure and annotation of the region can be found in Fig. 2. Te risk alleles on chromosome 22 are present in 40% of the cases, however when looking at the distribution of alleles for the chromosome 22 and chromosome 18 loci (at 49 Mb) then 96% of the cases carried at least one risk allele from one of the two loci versus only 70% of the controls. Te distribution of genotypes between cases and controls is shown in Figure S2. Association to age of onset. To investigate potential loci associated to early onset of pyometra, we per- formed an association analysis within the cases only, using age of onset in days as a continuous variable. No SNPs reached Bonferroni corrected signifcance (Fig. 1e, f). Two loci on chromosome 15 and 32 stood out and were considered as suggestively associated. Te most strongly associated SNP on chromosome 15 (chr15:59,440,763, p = 9.0 × 10–6) is located within intron 3 of the Nuclear Assembly Factor 1 ribonucleoprotein (NAF1) gene. Te most strongly associated SNP on chromosome 32 (chr32:22,285,412, p = 4.32 × 10 –5) was located in intron 1 of the Endomucin (EMCN) gene. Investigation of top locus identifes non‑synonymous SNP in the ABCC4 gene. Te genome- wide signifcant locus on chromosome 22 was defned as an 18.2 kb haplotype block of 5 GWAS SNPs in com- plete LD (r2 = 1.00, chr22:45,875,420–45,893,599 bp) spanning introns 18 and 19 of the ATP-binding cassette transporter 4 gene (ABCC4, ENSCAFG00000005433, ENSCAFT00000008769, UniProt F1PNA2), (Fig. 3a–c). Te allele frequency for the risk haplotype was 0.21 in the cases versus 0.05 in the controls (Table 1), resulting in an odds ratio of 4.8 (95% CI 2.3–9.9). A summary of the allele frequencies and p values for the fve GWAS SNPs is shown in Table 1. To further investigate the associated locus on chromosome 22, we generated whole genome sequencing data from a pool of 10 pyometra cases (22X mean coverage), which were all heterozygous for the associated risk haplotype on chromosome 22. In addition, sequencing data was generated from one individual homozygous for the GWAS risk haplotype (23X coverage) and 10 individually barcoded individuals homozygous for the non- risk alleles (3 cases and 7 controls; 4.4X mean coverage).
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